1. Field of the Invention
This invention relates to a method of preparing a commercial plasma preparation that will allow preparation of PT calibration curves with values about 100% and which will give results analogous to those obtained using fresh normal pooled plasma.
2. Description of the Related Art
The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using, e.g., coumadin. Thromboplastin reagents activate the "extrinsic" pathway of coagulation and are the basis for the PT test. Thromboplastin contains lipidated tissue factor (TF), which is the activator of the extrinsic pathway. This activation centers on Factor VII (FVII) and activated Factor VII (Factor VIIa), the TF-FVII Complex activates Factor X, which with Factor V activates Factor II to produce thrombin, which creates the fibrin clot.
There are several ways of expressing the results of the PT test. One system, the INR system, is recommended by the World Health Organization. However, many countries have not adopted this system for expressing PT results. Moreover, the INR system has only been validated for patients on oral anticoagulant control, but should not be used in expressing results from patients with other disease states, such as liver disease. Another system, commonly used in the United States, expresses the time in seconds for the blood to begin to coagulate. Still another system expresses the results in terms of a percentage PT ("% PT") which is read from a standard calibration (or dilution) curve prepared by diluting fresh normal pool plasma ("FNP") in 0.9% saline. (Other diluents work, but by convention, only saline is used.) The curve allows for the conversion of results from time in seconds to percent of normal activity (% PT). Unfortunately, in order for this system to be used, most laboratories have to prepare their own pool plasma and keep it frozen, usually in liquid nitrogen or frozen at -80.degree. C. Moreover, due to the inherent variation found in different plasma pools, there is no standardization between the plasma pools of different laboratories. Moreover, it has been shown that if a pool of plasma is prepared, the mean % PT value obtained from the pool is different than the mean % PT value obtained from the individual samples that were used to make the pool. It has also been shown that the collection of bulk collections of blood, as would be required to commercially prepare a lyophilized standard, causes a reduction in the measured % PT when compared with blood collected by venipuncture. See Important Differences Encountered in the Normal Plasma Pools used for the Control of Oral Anticoagulation. M. Burgess-Wilson, R. Burri and B. Woodhams, Thromb. Haemost. 69 Abstract 2081 (1993). Moreover, FNP cannot be sold until lyophilized. Lyophilization results in a plasma which, when reconstituted, has a % PT value lower than that found in a normal hospital pool of plasma. This reconstituted FNP is then used to prepare the standard curve. The dilutions usually used for the standard calibration curve are undiluted, 1:1, 1:2 and 1:4. Where reconstituted FNP is used, the undiluted sample is assigned a value of 100% PT. A PT assay is performed and the results (in seconds) are plotted on hyperbolic or reciprocal graph paper against the dilution (in %). See FIG. 1. Patient samples are tested undiluted and then read from this standard curve. However, using reconstituted FNP as a calibrator means that values for normal samples are above the top calibration point of the standard curve made using the reconstituted FNP. (By definition, 50% of all normal values would be above the top point of the standard curve.)
The % PT curve is not a straight line. Although a polynomial plot gives the most realistic curve through the data, many laboratories and users do not have the computer software required for such a procedure. Therefore, a linear curve through the points is commonly used. To make the results more accurate around the 100% region of the curve, the line is forced through the 100% point. One type of assay machine, the Medical Laboratory Automation ("MLA") Electra automated coagulometers, does not calculate % PT outside of certain ranges (above about 125% PT and below about 12% PT).
The recommended method of calculating % PT varies between the instrument manufacturers. There is no universally used standard procedure. Some instrument manufacturers, such as MLA, recommend forcing a straight line through 100%. Others recommend polynomial or non-forced straight lines. This introduces variability into the procedure, especially if the calibration plasma has a value of % PT much lower than 100%. See FIG. 2. In the examples that follow, the method of calculating the % PT was to use a forced linear curve through the 100% point using the SigmaPlot transformation.